Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of...
Reexamination Certificate
1998-08-14
2001-04-03
LeGuyader, John L. (Department: 1633)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
C435S320100, C435S252300, C536S023100, C536S023500
Reexamination Certificate
active
06210964
ABSTRACT:
FIELD OF THE INVENTION
The present invention is directed to an avian calcium-sensing receptor protein and to nucleic acid sequences encoding this protein. It encompasses assays that utilize the avian receptor and to transgenic animals that have been engineered to express mutated forms of the receptor. In addition, the invention is directed to methods for altering the activity of calcium receptors by administering small organic polyanions.
BACKGROUND OF THE INVENTION
An extracellular Ca
2+
-sensing receptor (CaR) has been cloned from both bovine and human parathyroids, and there is evidence indicating that this receptor plays a key role in regulating extracellular calcium homeostasis by controlling PTH secretion (Pollak, et al.,
Cell
75:1297-1303 (1993); Pollak, et al.,
Nature Genet.
8:303-307 (1994); Pollak, et al.,
J. Clin. Invest.
93:1108-1112 (1994)). The isolation and identification of the avian counterpart of this receptor and of polynucleotides encoding the receptor should aid in the development of methods for regulating serum calcium levels in chickens and related species. By increasing serum calcium in such animals, it is expected that more rapid growth should be obtainable due to an increased rate of bone deposition and that eggs of higher quality should be produced.
SUMMARY OF THE INVENTION
The present invention is based upon two related discoveries. First, a novel avian extracellular Ca
2+
-sensing receptor has been isolated and characterized. This receptor is structurally distinct from all similar receptors that have been identified and can be used in assays designed to identify agents that lead to an alteration in the serum calcium concentration of birds. Mutated forms of the receptor have been identified that have an altered sensitivity to extracellular calcium. These can be used in the development of transgenic chickens that have a higher than normal concentration of serum calcium and that have improved characteristics in terms of growth and egg production.
The second discovery forming the basis of the present invention is that small organic polyanions can be used to antagonize the interaction of calcium with receptor (“CaR”) in chickens and in other species as well. Thus, agents such as suramin may be used to treat patients that are suffering from abnormally low serum calcium concentrations. In addition, these agents may be included in the feed of farm animals (e.g., cattle, pigs, horses, sheep, chickens etc.) to increase the availability of serum calcium for bone deposition. This should lead to animals that are healthier and that grow at a faster rate. In the case of chickens, it should also lead to animals that produce eggs with stronger shells.
In its first aspect, the invention is directed to a substantially pure avian CaR having an amino hacid sequence consisting essentially of the sequence of SEQ ID NO:2 (see also FIGS.
2
A-
2
D). The term “consisting essentially of,” is meant to encompass proteins having exactly the same amino acid sequence as that shown in the sequence listing, as well as proteins with differences that are not substantial, as evidenced by their retaining the basic, qualitative functional properties of CaR. A “substantially pure” protein is one that has been separated from other accompanying biological components and will typically comprise at least 85% of a sample, with greater percentages being preferred. Many means are available for assessing the purity of a protein within a sample, including analysis by polyacrylamide gel electrophoresis, chromatography and analytical centrifugation.
The invention encompasses antibodies that bind specifically to avian CaR (i.e., that have at least a hundredfold greater affinity for avian CaR than for any protein that is not an extracellular calcium-sensing receptor) and antibodies made by a process involving the injection of a pharmaceutically acceptable preparation of CaR into an animal capable of antibody production. In a preferred embodiment, monoclonal antibody to avian CaR is produced by injecting the pharmaceutically acceptable preparation into a mouse and then fusing mouse spleen cells with myeloma cells.
The invention is also directed to a substantially pure polynucleotide encoding a protein consisting essentially of the amino acid sequence of SEQ ID NO:2, expression vectors comprising such polynucleotides, and host cells transformed with such vectors. Also included is the recombinant avian CaR receptor protein produced by host cells made in this manner. Preferably, the polynucleotide encoding the avian CaR has the nucleotide sequence of SEQ ID NO:1 (see also FIGS.
2
A-
2
D). It is also preferred that the vectors and host cells prepared for the expression of CaR use this particular polynucleotide.
In another aspect, the present invention is directed to a method for assaying a test compound for its ability to bind to the avian CaR. The method is performed by incubating a source of CaR with a ligand known to bind to the receptor and with the test compound. The CaR used in the assay should be substantially free of other types of Ca
2+
receptors, i.e., greater than about 90% of the Ca
2+
receptors present should correspond to CaR. Upon completion of incubation, the ability of the test compound to bind to CaR is determined by the extent to which ligand binding has been displaced. A preferred source of CaR for use in the assay is a cell transformed with a vector for expressing the receptor and comprising a polynucleotide encoding a protein consisting essentially of the amino acid sequence of SEQ ID NO:2. Instead of using cells in the assay, a membrane preparation can be prepared from the cells and this can be used as a source of CaR. Although not essential, the assay can be accompanied by a determination of whether test compounds affect the activity of a second messenger pathway. Preferably this is accomplished either by measuring a product of the activation of phospholipase C (typically inositol phosphate) or by measuring changes in cytosolic calcium levels. Assays of this type should help to determine whether a compound that modulates the activity of CaR is acting as an agonist or antagonist to Ca
2+
.
The invention is also directed to a method for assaying a test compound for its ability to alter the expression of the CaR gene. This method is performed by growing cells expressing CaR, and preferably substantially free of other Ca
2+
receptors, in the presence of the test compound. Cells are then collected and the expression of CaR is compared with expression in control cells grown under essentially identical conditions but in the absence of the test compound. In preferred embodiments, the cells expressing CaR are cells transformed with an expression vector comprising a polynucleotide sequence encoding a protein consisting essentially of the amino acid sequence of SEQ ID NO:2. Test compounds that may be used include oligonucleotides at least 15 nucleotides in length and comprising a sequence complementary to a sequence shown in SEQ ID NO:1. The preferred method for determining receptor expression is by means of Western blot analysis using CaR-specific antibody.
In another aspect, the present invention is directed to a DNA construct that can be used in the development of transgenic animals expressing a mutated form of CaR. The construct should contain a targeting segment that consists essentially of the nucleotide sequence encoding the wild type avian CaR gene, as shown SEQ ID NO:2, but in which one or more nucleotides in codon 127 have been modified. It has been found that mutations in this codon result in receptors that have an altered sensitivity to Ca
2+
. Preferred mutations for reducing the sensitivity of CaR to extracellular calcium are those that result in codon 127 encoding tryptophan. Preferred mutations for increasing the sensitivity of CaR to extracellular calcium are those that result in codon 127 encoding lysine, asparagine, valine, glutamine, and alanine. The targeting segment encoding the mutated form of CaR is operably linked to a pro
Bai Mei
Brown Edward M.
Diaz Ruben
Quinn Stephen J.
LeGuyader John L.
Pillsbury & Winthrop LLP
Sanzo Michael A.
The Brigham and Women's Hospital Inc.
Wilson Michael C.
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